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Abstract
Background
HLA-G is a non-classical MHC class I molecule which exerts strong immunosuppressive effects on various immune cells. Several membrane-bound and soluble isoforms are known. Physiologically, HLA-G is predominantly expressed in the placenta, where it contributes to protecting the semi-allogeneic embryo from rejection by the maternal immune system. However, HLA-G is also often upregulated during tumourigenesis, such as in ovarian cancer. The aim of this thesis is to investigate how soluble HLA-G may contribute to local immunosuppression in ovarian carcinomas, and to characterize HLA-G expression in different ovarian carcinoma subtypes and metastases.
Results
As reported by others, physiological HLA-G expression is restricted to few tissues, such as placenta and testes. Here, HLA-G was also detected in the medulla of the adrenal gland. In contrast, HLA-G expression was frequently detected in tumours of all assessed subtypes of ovarian carcinomas (serous, mucinous, endometrioid and clear cell). Highest expression levels were detected in high-grade serous carcinomas. In primary tumours, expression of HLA-G correlated with expression of classical MHC class I molecules HLA-A, -B and -C. Surprisingly, high levels of HLA-G were also detected on dendritic cells in local lymph nodes. As no expression of HLA-G was inducible in monocytes or dendritic cells from healthy donors in response to IL-10 or IL-4, we speculated that tumour-derived soluble HLA-G might be transferred to dendritic cells via the lymphatic system. Accordingly, high levels of tumour-derived soluble HLA-G were detected in ovarian cancer ascites samples. In vitro, dendritic cells expanded in the presence of IL-4, IL-10 and GM-CSF (DC-10) were particularly prone to binding high amounts of soluble HLA-G via ILT receptors. Furthermore, HLA-G loaded DC-10 cells inhibited the proliferation of CD8 effector cells and induced regulatory T cells, even when the DC-10 cells had been fixed with paraformaldehyde.
Conclusion
The immunosuppressive molecule HLA-G is overexpressed in high-grade serous ovarian carcinomas, which account for the majority of ovarian cancers. In particular tumours with a high mutational burden and intact expression of classical, immunogenic MHC class Ia molecules may use HLA-G to escape from immunosurveillance. Additionally, tumour-derived soluble HLA-G may inhibit adaptive immune responses by binding to dendritic cells in local lymph nodes. Dendritic cells usually play a decisive role in the initiation of adaptive anti-tumour immune responses by presenting tumour antigens to cytotoxic T cells. In contrast, dendritic cells loaded with soluble HLA-G inhibit the proliferation of effector T cells and promote the induction of regulatory T cells. Thus, soluble HLA-G that is transferred to dendritic cells via lymphatic vessels may enable ovarian carcinomas to remotely suppress anti-tumour immune responses in local lymph nodes. This novel immune-escape mechanism may also exist in other solid tumours that express HLA-G.
The diploid, polymorphic yeast Candida albicans is one of the most important human pathogenic fungi. C. albicans can grow, proliferate and coexist as a commensal on or within the human host for a long time. However, alterations in the host environment can render C. albicans virulent. In this review, we describe the immunological cross-talk between C. albicans and the human innate immune system. We give an overview in form of pairs of human defense strategies including immunological mechanisms as well as general stressors such as nutrient limitation, pH, fever etc. and the corresponding fungal response and evasion mechanisms. Furthermore, Computational Systems Biology approaches to model and investigate these complex interactions are highlighted with a special focus on game-theoretical methods and agent-based models. An outlook on interesting questions to be tackled by Systems Biology regarding entangled defense and evasion mechanisms is given.
Hintergrund: Das Absterben Neuromelanin (NM)-haltiger Zellen in der substantia nigra (SN), und die daraus resultierende Erniedrigung des Dopaminspiegels im striatum, ist ein pathologisches Hauptmerkmal der Parkinsonschen Krankheit. Ein neuerlicher Nachweis von Anti-Melanin-Antikörpern gibt Anlass zur Vermutung, dass NM ein Autoantigen sein könnte. In dieser Arbeit wurde gezeigt, dass NM tatsächlich von dendritischen Zellen (DZ), die in vivo hauptverantwortlich für die Auslösung von T- und B-Zellantworten sind, erkannt wird. Die Erkennung von NM durch DZ ist eine unabdingbare Voraussetzung für die Einleitung einer adaptiven Immunantwort. Methoden: Murine dendritische Zellen (mDZ) wurden aus Knochenmarkszellen generiert und mit NM aus humaner SN oder synthetischem Dopaminmelanin (DAM) behandelt, nachdem beide Melanine endotoxinfrei getestet wurden. Die Phagozytose von NM wurde mittels konfokaler Mikroskopie dokumentiert. Die Expression von MHC II und CD86 wurde mittels Durchflusszytometrie (FACS) analysiert. Zytokinkonzentrationen von TNF- und dem Interleukin IL-6 wurden mit ELISA-Assays bestimmt. Abschließend wurde die Funktion der durch NM aktivierten DZ mit einer allogenen mixed lymphocyte reaction (MLR) überprüft. Ergebnisse: NM wurde von den mDZ effektiv phagozytiert, woraufhin die mDZ einen reifen Phenotyp (CD86high/MHC IIhigh) zeigten. Zusätzlich sekretierten durch NM aktivierte mDZ die Zytokine IL-6 and TNF-. Schließlich ließen die mDZ T-Zellen in einer MLR proliferieren, und beweisen so ihre Funktionalität und die Fähigkeit eine primäre T-Zellantwort auszulösen. Im Gegenteil dazu konnte DAM, dem die Protein- und Lipidkomponenten von NM fehlen und nur das Melaninrückrat mit NM gemeinsam hat, nur einen kleinen Effekt bei den mDZ hervorrufen. Diskussion: NM wird von DZ in vitro erkannt und bewirkt deren Reifung. Sollte der Vorgang auch in vivo stattfinden, besteht die Möglichkeit, dass SN-Antigene dem adaptiven Immunsystem präsentiert werden, was in einzelnen Fällen zur Einleitung einer adaptiven Immunantwort führen könnte. NM könnte also der Auslöser für einen autoimmunen Pathomechanismus in der parkinsonschen Krankheit sein.
Investigations of Measles virus regulation on activation and function of antigen presenting cells
(2008)
Interaction with dendritic cells (DCs) is considered as central to immunosuppression induced by viruses, including measles virus (MV). Commonly, viral infection of DCs abrogates their ability to promote T cell expansion, yet underlying mechanisms at a cellular level are undefined. It appears that MV-WTF infection modulate DCs morphology and dynamic adhesion on extra cellular matrix proteins such as FN or ICAM-1. By morphological criteria, WTF-DCs resembled LPS-DCs, associated with their mature phenotype also adhered less efficiently to the FN or ICAM-1 support. Reduced adhesion could not be explained by a lack of 1-integrin expression or activation. Similarly, MV-DCs strongly resembled LPS-DCs in that levels of focal adhesion kinase phosphorylated at Y397 were high and not further enhanced upon FN ligation. Fascin, a downstream effector of integrin signaling was highly upregulated in LPS-DCs and moderately in WTF-DCs, and differences in its subcellular distribution were not observed between both cell cultures. Apparently, however, fascin associated less efficiently with PKC in WTF-DCs then in LPS-DCs. In line with findings for murine DCs, high motility of mature human DCs was found to require expression of Rac-GTPases. Human LPS-DCs and more so, DC transfected to express constitutively active Rac1 were the most motile DC-species analysed, confirming that migration of human DC also involved Rac activity. The velocity of WTF-DCs on FN is below that of LPS-DCs, indicating that maturation induced by WTF may be insufficient to completely promote integrin signaling which leads to Rac activation. The organisation of MV-DC/T cell interfaces was consistent with that of functional immune synapses with regard to CD3 clustering, MHC class II surface recruitment and MTOC location. These analyses are based in the selection of stable conjugates. Subsequently, however, neither contacts nor calcium flux can be stabilised and sustained in the majority of MV-DC/T cell conjugates and only promoted abortive T cell activation. Formation of spatially organised IS in T cells requites, prolonged contact durations. Therefore, aberrant distribution patterns of CD3 in these structures, if occurring, are not likely to contribute to the type of contacts predominating for WTF-DC/T cell interactions. It is also likely that transient interactions of less than 2 minutes may if at all, not efficiently support viral transmission to T cells. Transient interactions are typically observed with immature DCs in the absence of antigen, but this is not likely to be relevant in our allogenic system, which includes SA-loaded WTF-DCs. Thus, MV-infected DCs retain activities required for initiating, but not sustaining T cell conjugation and activation. This is partially rescued if surface expression of the MV glycoproteins on DCs is abolished by infection with a recombinant MV encoding VSV G protein instead, indicating that these contribute directly to synapse destabilisation and thereby act as effectors of T cell inhibition.
Dendritic cells (DCs) are antigen presenting cells which serve as a passage between the innate and the acquired immunity. Aspergillosis is a major lethal condition in immunocompromised patients caused by the adaptable saprophytic fungus Aspergillus fumigatus. The healthy human immune system is capable to ward off A. fumigatus infections however immune-deficient patients are highly vulnerable to invasive aspergillosis. A. fumigatus can persist during infection due to its ability to survive the immune response of human DCs. Therefore, the study of the metabolism specific to the context of infection may allow us to gain insight into the adaptation strategies of both the pathogen and the immune cells. We established a metabolic model of A. fumigatus central metabolism during infection of DCs and calculated the metabolic pathway (elementary modes; EMs). Transcriptome data were used to identify pathways activated when A. fumigatus is challenged with DCs. In particular, amino acid metabolic pathways, alternative carbon metabolic pathways and stress regulating enzymes were found to be active. Metabolic flux modeling identified further active enzymes such as alcohol dehydrogenase, inositol oxygenase and GTP cyclohydrolase participating in different stress responses in A. fumigatus. These were further validated by qRT-PCR from RNA extracted under these different conditions. For DCs, we outlined the activation of metabolic pathways in response to the confrontation with A. fumigatus. We found the fatty acid metabolism plays a crucial role, along with other metabolic changes. The gene expression data and their analysis illuminate additional regulatory pathways activated in the DCs apart from interleukin regulation. In particular, Toll-like receptor signaling, NOD-like receptor signaling and RIG-I-like receptor signaling were active pathways. Moreover, we identified subnetworks and several novel key regulators such as UBC, EGFR, and CUL3 of DCs to be activated in response to A. fumigatus. In conclusion, we analyze the metabolic and regulatory responses of A. fumigatus and DCs when confronted with each other.